Replication of the HIV-1 genome, like all nucleic acids, involves synthesis of a minus and a plus strand. Despite the application of quantitative real time PCR technology, several questions regarding the complex nature of reverse transcription remain unanswered, including the rates of RNA- and DNA-dependent DNA synthesis during infection. Currently available PCR methods cannot distinguish between the two strands. Because reverse transcription occurs of both strands simultaneously, and has the potential to copy the same minus-strand multiple times through displacement synthesis, it is not possible to analyze the kinetics of reverse transcription using conventional PCR. To determine the rates of HIV-1 reverse transcription in infected cells, we have developed a novel SSA assay using single-stranded padlock probes that are specifically hybridized to either the minus strand or the plus strand, ligated, and quantified using real-time PCR. Using SSA, we have determined for the first time the rates of HIV-1 RNA-dependent DNA synthesis in 293T and human primary CD4+ T cells. The results showed that the rates of minus-strand DNA synthesis in 293T cells and in activated CD4+ T cells were essentially identical (68 nt/min). We also determined the rates of minus-strand DNA transfer (4 min), plus-strand DNA transfer (28 min), and initiation of plus-strand DNA synthesis (9 min) in 293T cells. Plus-strand-specific products that were separated by 3- to 4-kb accumulated with very similar kinetics; as a result it was not possible to measure the rate of plus-strand DNA synthesis. These results indicated that plus-strand DNA synthesis is initiated at multiple sites. We also determined whether inhibitors of RT (AZT, d4T, ddI, and EFV) displayed differential effects during minus- and plus-strand DNA synthesis. The results showed that RNA-dependent DNA synthesis was substantially inhibited by AZT and d4T, moderately inhibited by EFV, and minimally inhibited by ddI. The observation that AZT and d4T inhibited minus-strand DNA synthesis by 95% at concentrations that inhibited viral replication by 95% suggests that AZT and d4T inhibit reverse transcription primarily during minus-strand DNA synthesis. EFV and ddI appear to have a greater inhibitory effect during plus-strand synthesis. These studies have generated a novel strand-specific PCR technique that should be widely applicable to a variety of molecular studies, and provide the first measurements of several key steps during HIV-1 reverse transcription in cells. [Corresponds to Pathak Project 3 in the April 2007 site visit report of the HIV Drug Resistance Program]